Novel antimicrobial activity of gemfibrozil

ABSTRACT

The present invention provides for a method for inhibiting growth of a bacterium which consists essentially of contacting the bacterium with a compound having the structure:  
                 
 
     wherein each of R 1 , R 2 , R 3 , R 4 , R 5  and R 6  may be independently H, F, Cl, Br, I, —OH, —OR 7 , —CN, —COR 7 , —SR 7 , —N(R 7 ) 2 , —NR 7 COR 8 , —NO 2 , —(CH 2 ) p OR 7 , —(CH 2 ) p X(R 7 ) 2 , —(CH 2 ) p XR 7 COR 8 , a straight chain or branched, substituted or unsubstituted C 1 -C 10  alkyl, C 2 -C 10  alkenyl, C 2 -C 10  alkynyl, C 3 -C 10  cycloalkyl, C 3 -C 10  cycloalkenyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein a linkage to the benzene ring may alternatively be —N—, —S—, —O— or —C—; wherein R 7  or R 8  may be independently H, F, Cl, Br, I, —OH, —CN, —COH, —SH 2 , —NH 2 , —NHCOH, —(CH 2 ) p OH, —(CH 2 ) p X(CH 2 ), —(CH 2 ) p XCOH, a straight chain or branched, substituted or unsubstituted C 1 -C 10  alkyl, thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl; wherein A may be —N 2 —, —NH—, —C═C═CH 2 —, —C≡C—C 2 HOH—, —C≡C—CH 2 —, —CH 2 —CH 2 —O—, —CH 2 —CH 2 —CH 2 —O—, —S—, —S(═O) 2 —, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—; and wherein Q, p, N and X may independently be an integer from 1 to 10, or if Q is 1 A may be a (C 1 -C 10 )-alkyl chain, (C 1 -C 10 )-alkenyl chain or (C 1 -C 10 )-alkynyl chain which can optionally be interrupted 1 to 3 times by —O— or —S— or —N—; or a pharmaceutically acceptable salt or ester thereof, which compound is present in a concentration effective to inhibit growth of the bacterium. A may be an (C 1 -C 10 )-alkylene chain, (C 1 -C 10 )-alkyl chain, or (C 1 -C 10 )-alkynyl chain which is branched or unbranched, substituted or unsubstituted and can optionally be interrupted 1 to 3 times by —O— or —S— or —N—.

[0001] The invention disclosed herein was made with Government supportunder Grant No. AI23549 and AI20516 from NIAID. Accordingly, the U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0002] Throughout this application, various publications are referencedby author and date. Full citations for these publications may be foundlisted alphabetically at the end of the specification immediatelypreceding Sequence Listing and the claims. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

[0003] Gemfibrozil (GFZ) is a compound that has been utilized as a drugfor increasing intracellular accumulation of hydrophilic anionic agents(U.S. Pat. No. 5,422,372, issued Jun. 6, 1995) and as a lipid regulatingcomposition (U.S. Pat. No. 4,859,703, issued Aug. 22, 1989). Gemfibrozilhas been shown to be effective in increasing the amount of cholesterolexcreted in to bile. (Ottmar Leiss et al., Metabolism, 34(1):74-82(1985)). Gemfibrozil is described in U.S. Pat. No. 3,674,836 and in TheMerck Index, 11 ed., Merck & Co., Inc. Rahway, N.J. 1989; #4280.Gemfibrozil, a drug which therapeutically lowers triglycerides andraises HDL-cholesterol levels, previously has not been reported to haveantimicrobial activity. (Brown, 1987; Oliver et al., 1978 and Palmer etal., 1978)

SUMMARY OF THE INVENTION

[0004] The present invention provides for a method for. inhibitinggrowth of a bacterium which consists essentially of contacting thebacterium with a compound having the structure

[0005] In the compound each of R₁, R₂, R₃, R₄, R₅ and R₆ may beindependently H, F, Cl, Br, I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂,—NR₇COR₈, —NO₂, —(CH₂)_(p)OR₇, —(CH₂)_(p)X(R₇)₂, —(CH₂)_(p)XR₇COR₈, astraight chain or branched, substituted or unsubstituted C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, orheteroaryl; wherein R₇ or R₈ may be independently H, F, Cl, Br, I, —OH,—CN, —COH, —SH₂, —NH₂, —NHCOH, —(CH₂)_(p)OH, —(CH₂)_(p)X(CH₂),—(CH₂)_(p)XCOH, a straight chain or branched, substituted orunsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl,phenyl, substituted phenyl, or heteroaryl; wherein A may be —N₂—, —NH—,—C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—C₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—, —S—,—S(═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—; and wherein Q, p, N and Xmay independently be an integer from 1 to 10, or if Q is 1 A may be a(C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chainwhich is branched or unbranched, substituted or unsubstituted and canoptionally be interrupted 1 to 3 times by —O— or —S— or —N—; or apharmaceutically acceptable salt or ester thereof, which compound ispresent in a concentration effective to inhibit growth of the bacterium.In this method, A may be an (C₁-C₁₀)-alkylene chain, (C₁-C₁₀)-alkylchain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chain which isbranched or unbranched, substituted or unsubstituted and can optionallybe interrupted 1 to 3 times by —O— or —S— or —N—; and wherein the etherlinkage to the benzene ring may be alternatively —S—, —N— or —C—.

BRIEF DESCRIPTION OF THE FIGURES

[0006]FIG. 1. MICs (minimal inhibitory concentration) for gemfibrozilwere determined by incubating L. pneumophila or F4b with variousconcentrations of GFZ. in AYE broth (microbiological media). Bacteriawere present at an initial concentration of 1×10⁶ CFU's (colony formingunits)/ml. Growth was turbidimetrically assessed by determining the ODat 600 nm after a 48 hour incubation at 37° C.

[0007]FIG. 2. MICs for probenecid were determined by incubating L.pneumophila, resuspended to 1×10⁶ CFU's/ml; with various concentrationsof probenecid in AYE broth. Growth was turbidimetrically assessed bydetermining the OD at 600 nm after 48 hours at 37° C.

[0008]FIG. 3. MICs for clofibric acid were determined by incubating L.pneumophila, resuspended to 1×10⁶ CFU's/ml, with various concentrationsof clofibric acid in AYE broth. Growth was turbidimetrically assessed bydetermining the OD at 600 nm after 48 hours at 37° C.

[0009]FIG. 4. Bacteria were screened for sensitivity to gemfibrozilusing a zone of inhibition assay. The assay was performed by addingbacteria to a suitable nutrient agar plate, adding a disk. containinggemfibrozil to the plate, and then incubating the plate at theappropriate temperature. The presence of a zone of inhibition (areaaround the disk where no growth occurred) was considered positive forsensitivity.

[0010]FIG. 5. Twenty one clinical and CDC M. tuberculosis strains,demonstrating different drug resistant profiles, were tested forsensitivity to gemfibrozil. Disks containing a given amount of GFZ wereadded to each of four quadrants of a plate. Five mls of Middlebrook agarwere added to each quadrant, and the drug was allowed to diffusethroughout the agar in each quadrant overnight. 100 μls of a standarddilution of each M. tuberculosis strain were added to each quadrant, andthe plates were incubated for three weeks at 37° C. No growth wasindicated by (−). Fewer than 50 colonies, were counted; (+) 50-100colonies; (++) 100-200 colonies; (+++) 200-500 colonies; (++++)confluent growth.

[0011]FIG. 6A-6B. GFZ induces large distending inclusions in asubpopulation of L. pneumophila grown in the presence of a subinhibitoryconcentration of GFZ. (A) Stationary phase L. pneumophila, grown in AYE,stained with Nile Blue A. Numerous nondistending granules present in themajority of the bacteria. (B) Stationary phase L. pneumophila, grown inAYE(+GFZ), stained with Nile Blue A. Numerous large, distending granulespresent in a subpopulation of the bacteria, other bacteria demonstratefew to no inclusions.

[0012]FIG. 7. Electron micrograph, 20,000×, of L. pneumophila grown tolog phase on a CYE plate. Note the presence of small, non-distendinginclusions.

[0013]FIG. 8. Electron micrograph, 20,000×, of L. pneumophila grown on aCYE plate containing an inhibitory concentration of GFZ. Note thepresence of large, distending inclusions in a subpopulation of thebacteria, and the absence of inclusions in other bacteria.

[0014]FIGS. 9A, 9B, 9C and 9D. Demonstration of an intermediatephenotype during GFZ-induced inclusion development in L. pneumophila.Electron micrographs, 8,000×, of pelleted L. pneumophila and F4b grownin AYE broth in the presence or absence of GFZ 85 μg/ml for 4.5 hours.L. pneumophila demonstrates increased numbers of inclusions, while F4b,the GFZ semi-resistant mutant, does not. (A) L. pneumophila; no GFZ, (B)F4b; no GFZ (C) L. pneumophila; GFZ 85 μg/ml (D) F4b; GFZ 85 μg/ml.

[0015]FIGS. 10A, 10B, 10C and 10D. Fatty acid compositions of wild typeL. pneumophila, and the GFZ semi-resistant mutant F4b, grown in thepresence or absence of a subinhibitory concentration of gemfibrozil.Fatty acid compositions were assessed by saponifying, methylating, andextracting the fatty acids present in the bacteria scraped from theplates, and then injecting the methylated fatty acids into a gaschromatograph. A step temperature program was used such that as thetemperature was increased, sequentially longer chain fatty acids werereleased from the column and detected as peaks on the chromatogram. (A)Wild type L. pneumophila grown on CYE plates in the absence of GFZ (B)Wild type L. pneumophila grown-on CYE(GFZ 30 μg/ml) plates; peaks thathave decreased in size are marked by arrows, new peaks are marked bydots. (C) F4b grown on CYE plates in the absence of GFZ (D) F4b grown onCYE(GFZ 30 μg/ml) plates.

[0016]FIG. 11. Sensitivity of L. pneumophila and F4b to INH. Bacterialoverlays on CYE agar plates were prepared by adding 2×10⁷ bacteria to 3mls of melted 50° C. agar and pouring the mixture over 15 ml CYE agarplates. Sterile disks containing 1 mg of INH, or 250 μg of GFZ wereadded to the overlays, and the plates were incubated for four days.Sensitivity was assessed by measuring the diameter of the zone ofinhibition, the area where bacterial growth was inhibited, surroundingthe drug disks.

[0017]FIG. 12. Demonstration of inverse relationship between GFZsensitivity and INH sensitivity using INH-resistant. F4b revertants.INH-resistant F4b revertants were obtained by adding F4b to CYE-INH drugplates (400 μg/ml) and screening for spontaneous INH-resistant mutantsafter four days of incubation at 37° C. INH resistant colonies, whicharose at a frequency of 1/10⁻⁷, were picked, passed non-selectivelythree times on CYE, and then tested for GFZ and INH sensitivity usingthe zone of inhibition assay. The assay was performed by adding, 2×10⁷bacteria to 3 mls of melted 55° C. agar, pouring the mixture over 15 mlCYE plates, and then adding 1 mg INH sterile disks and 250 μg GFZsterile disks to the overlays. After a four day incubation at 37° C.,the, diameter of the zones of inhibition were measured. The coloniesindicated by the left bar under the histogram retained the parentalphenotype and thus were not revertants. The colonies indicated by theright bar under the histogram regained GFZ sensitivity as INHsensitivity was lost.

[0018]FIG. 13. Sensitivity of L. pneumophila and F4b to ethionamide.Ethionamide resistance correlates with GFZ resistance in the L.pneumophila-derived mutant F4b. Sensitivity was assessed by measuringthe diameter of the zone of inhibition in bacterial overlays surrounding250 μg GFZ disks, or 500 μg ethionamide disks.

[0019]FIGS. 14A-14B. GFZ inhibits intracellar multiplication of L.pneumophila in PMA-differentiated HL-60 cells. (A) Monolayers ofPMA-differentiated HL-60 cells were infected for 2.5 hours with L.pneumophila or F4b in the wells of 96 well microtiter plates. The wellswere washed to remove extracellular bacteria, and medium containing 100μg/ml of GFZ was added to the monolayers. Bacteria were titered atdifferent time points by lysing the monolayers and counting the totalnumber of CFU's present in the lysate and medium. (B) Monolayers ofPMA-differentiated HL-60 cells were infected with increasingconcentrations of L. pneumophila in the wells of 96 well microtiterplates. After 2.5 hours GFZ was added to the wells to a finalconcentration of 100 μg/ml. An MTT assay was performed after a five dayincubation at 37° C. to assess HL-60 cell viability. Reduction of MTT byviable HL-60 cells was measured spectrophotometrically at 590 nm.

[0020]FIGS. 15A, 15B and 15C. GFZ inhibits intracellular multiplicationof L. pneumophila in monocytic cells. (A) Monolayers of human peripheralblood derived monocytes were infected with L. pneumophila in the wellsof 96 well microtiter plates. After 2.5 hours, the well were-washed andmedium containing GFZ 100 μg/ml was added to the monolayers. Bacteriawere titered at different time points by lysing the monolayers andcounting the total number of CFU's present in the lystate and medium ofeach well. (B) Monolayers of human peripheral blood derived macrophageswere infected with F4b and titered for CFU's as described above. (C)Monolayers of the murine macrophage J774 cell line were infected with L.pneumophila and titered for CFU's as described above.

[0021]FIG. 16. A L. pneumophila 2.1 kb DNA insert, expressed from pBSK,complements the envM E.coli ts mutant and confers sensitivity to GFZ atthe restrictive temperature, 42° C., on low osmolarity LB plates. tsenvM E.coli containing pBSK:2.1 were grown overnight in the presence ofampicillin, and then diluted 10⁻². 100 μl of this dilution was mixedwith 3 mls of melted 55° C. agar and poured over low osmolarity LBplates. Disks containing 5 mg of GFZ were added to the overlays, and theplates were incubated at 30° C. or 42° C. overnight. The diameter of thezones were measured to assess GFZ sensitivity.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides for a method for inhibiting growthof a bacterium which consists essentially of contacting the bacteriumwith a compound having the structure

[0023] wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ may be independently H,F, Cl, Br, I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇COR₈, —NO₂,—(CH₂)_(p)OR₇, —(CH₂)_(p)X(R₇)₂, —(CH₂)_(p)XR₇COR₈, a straight chain orbranched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl,methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;wherein R₇ or R₈ may be independently H, F, Cl, Br, I, —OH, —CN, —COH,—SH₂, —NH₂, —NHCOH, —CH₂)_(p)OH, —(CH₂)_(p)X(CH₂), —(CH₂)_(p)XCOH, astraight chain or branched, substituted or unsubstituted C₁-C₁₀ alkyl,C₂-C₁₀ alkenyl; C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, orheteroaryl; wherein A may be —N₂—, —NH—, —C═C═CH₂—, —C≡C—C₂HOH—,—C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—, —S—, —S(═O)₂—, —C═O—, —C═O—O—,—NH—C═O—, —C═O—NH—; and wherein Q, p, N and X may independently be aninteger from 1 to 10, or if Q is 1 A may be a (C₁-C₁₀)-alkyl chain,(C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chain which is branched orunbranched, substituted or unsubstituted and can optionally beinterrupted 1 to 3 times by —O— or —S— or —N—; or a pharmaceuticallyacceptable salt or ester thereof, which compound is present in aconcentration effective to inhibit growth of the bacterium.

[0024] In this method, A may be an (C₁-C₁₀)-alkylene chain, (C_(1-C)₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chain whichis branched or unbranched, substituted or unsubstituted and canoptionally be interrupted 1 to 3 times by —O— or —S— or —N—. The etherlinkage to the benzene ring may alternatively be —N—, —S— or —C—.

[0025] In one embodiment, the compound may include the following:

[0026] R₁=R₄=CH₃ or —OH,

[0027] R₂=R₃=R₅=R₆=H or —OH,

[0028] A=CH₂,

[0029] and Q=3.

[0030] In one embodiment, the compound may include the following:

[0031] R₃=Cl,

[0032] R₁=R₂=R₄=R₅=R₆=—OH or H,

[0033] and Q=0.

[0034] In anther embodiment, the compound may include:

[0035] R₆=CH(CH₃)₂,

[0036] R₁=R₂=R₄=R₅=H or —OH,

[0037] and Q=0.

[0038] In another embodiment, the compound may include:

[0039] R₃=Cl,

[0040] R₆=C₂H₅,

[0041] R₁=R₂=R₄=R₅=H or —OH,

[0042] and Q=0.

[0043] The bacterium may include Legionella pneumophila, Mycobacteriumtuberculosis, Bacillus subtilis, Bacillus Megaterium, PseudomonasOleovorans, Alcaligenes eutrophus, Rhodococcus sp., Citrobacter freundi,Group A Streptococcus sp., Coag neg Staphylococcus aureus or Nocardiasp. The bacterium may be Legionella pneumophila. The bacterium may beMycobacterium tuberculosis. The bacterium may be Nocardia sp. Thebacterium may be in a eukaryotic cell.

[0044] The concentration of the compound may be from about 5 μg/ml toabout 100 μg/ml. In another embodiment, the concentration of thecompound may be 20 μg/ml.

[0045] The present invention also provides a method for alleviating thesymptoms of a bacterial infection in a subject which consistsessentially of administering to the subject an amount of a compoundhaving the structure

[0046] wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ are as defined above.The ether linkage to the benzene ring may alternatively be —N—, —S— or—C—.

[0047] The method also includes use of a pharmaceutically acceptablesalt or ester thereof, which compound is present in a concentrationeffective to inhibit bacterial growth and thus alleviate the symptoms ofthe bacterial infection in the subject.

[0048] The bacterial infection may be associated with a bacterium listedabove. The subject may be a human or an animal. The bacterial infectionmay be associated with Leprosy, Brucella or Salmonella. Theconcentration of the compound may be from about 5 μg/ml blood of thesubject to about 180 μg/ml blood of the subject. In one embodiment, theconcentration of the compound may be 90 μg/ml blood of the subject. Theadministration to the subject may be oral.

[0049] The present invention also provides a method of inhibitingactivity of Enoyl Reductase Enzyme which includes contacting the enzymewith a compound having the structure

[0050] wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ are as defined above.The ether linkage to the benzene ring may alternatively be —N—, —S— or—C—.

[0051] As used herein Enoyl Reductase Enzyme includes enzymes havingenoyl reductase activity. Such enzymes may be bacterial enoyl reductasesor eukaryotic enoyl reductases. Examples of bacterial enoyl reductasesinclude those from the bacterium listed above. The enoyl reductase maybe one of the enoyl reductases from L. Pneumophila. The enoyl reductasemay be a gene product of a gene that hybridizes with moderate or highstringency with the envM gene.

[0052] The enzyme may be in a bacterium. The bacterium may be Legionellapneumophila, Mycobacterium tuberculosis, Bacillus subtilis, BacillusMegaterium, Pseudomonas Oleovorans, Alcaligenes eutrophus, Rhodococcussp., Citrobacter freundi, Group A Streptococcus sp., Coag negStaphylococcus aureus or Nocardia sp. The bacterium may be Legionellapneumophila. The bacterium may be Mycobacterium tuberculosis. The enzymemay be in a cell. The cell may be a mammalian cell. The concentration ofthe compound may be from about 5 μg/ml to about 100 μg/ml. Theconcentration of the compound may be 20 μg/ml.

[0053] The present invention provides for a method of altering a pathwayof fatty acid synthesis in a bacterium which comprises contacting thebacteria with a compound having the structure

[0054] wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ is as defined above.The ether linkage to the benzene ring may alternatively be —N—, —S— or—C—.

[0055] The present invention provides for a method for determiningwhether or not a bacterium is sensitive to a compound having thestructure

[0056] wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ is as defined above.The ether linkage to the benzene ring may alternatively be —N—, —S— or—C—.

[0057] The present invention provides for a method of selecting acompound which is capable of inhibiting the enzymatic activity of enoylreductase which includes: (A) contacting enoyl reductase with thecompound; (B) measuring the enzymatic activity of the enoyl reductase ofstep (A) compared with the enzymatic activity of enoyl reductase in theabsence of the compound, thereby selecting a compound which is capableof inhibiting the enzymatic activity of enoyl reductase. The compoundmay contact enoyl reductase at same site at which gemfibrozil contactsenoyl reductase. U.S. Pat. No. 5,422,372 discloses a method ofincreasing intracellular accumulation of hydrophilic anionic agentsusing gemfibrizol (gemfibrozil). U.S. Pat. No. 4,859,703 discloses lipidregulating compositions. U.S. Pat. No. 4,891,220 discloses a method andcomposition for treating hyperlipidemia. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

[0058] Another embodiment of the present invention is a kit which iscapable of detecting the presence of a particular organism based on thesensitivity of the organism to gemfibrozil. The present inventionprovides for a kit for detecting the presence of one or more organismsin a sample which comprises: (a) an agar or solution medium suitable forgrowth of the organism; (b) a means for testing growth of each organismin the presence and absence of gemfibrizol such that the growth of theorganism or lack thereof can be detected; (c) a means for determiningthe growth of the organism thus detecting the presence of one or moreorganisms in a sample. The kit may be in form of an assay, a screeningkit or a detection kit.

[0059] In one embodiment the compound of the present invention isassociated with a pharmaceutical carrier which includes a pharmaceuticalcomposition. The pharmaceutical carrier may be a liquid and thepharmaceutical composition would be in the form of a solution. Inanother embodiment, the pharmaceutically acceptable carrier is a solidand the composition is in the form of a powder or tablet. In a furtherembodiment, the pharmaceutical carrier is a gel and the composition isin the form of a suppository or cream. In a further embodiment theactive ingredient may be formulated as a part of a pharmaceuticallyacceptable transdermal patch.

[0060] A solid carrier can include one or more substances which may alsoact as flavoring agents, lubricants, solubilizers, suspending agents,fillers, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in, the shape and size desired. The powdersand tablets preferably contain up to 99% g of the active ingredient.Suitable solid carriers include, for example, calcium phosphate,magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin,cellulose, polyvinylpyrrolidine, low melting waxes and ion exchangeresins.

[0061] Liquid carriers are used in preparing solutions, suspensions,emulsions, syrups, elixirs and pressurized compositions. The activeingredient can be dissolved or suspended in a pharmaceuticallyacceptable liquid carrier such as water, an organic solvent, a mixtureof both or pharmaceutically acceptable oils or fats. The liquid carriercan contain other suitable pharmaceutical additives such assolubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoringagents, suspending agents, thickening agents, colors, viscosityregulators, stabilizers or osmo-regulators. Suitable examples of liquidcarriers for oral and parenteral administration include water (partiallycontaining additives as above, e.g. cellulose derivatives, preferablysodium carboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

[0062] Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by for example, intramuscular, intrathecal,epidural, intraperitoneal or subcutaneous injection. Sterile solutionscan also be administered intravenously. The active ingredient may beprepared as a sterile solid composition which may be dissolved orsuspended at the time of administration using sterile water, saline, orother appropriate sterile injectable medium. Carriers are intended toinclude necessary and inert binders, suspending agents, lubricants,flavorants, sweeteners, preservatives, dyes, and coatings. The activeingredient can be administered orally in the form of a sterile solutionor-suspension containing other solutes or suspending agents, forexample, enough saline or glucose to make the solution isotonic, bilesalts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleateesters of sorbitol and its anhydrides copolymerized with ethylene oxide)and the like. The active ingredient can also be administered orallyeither in liquid or solid composition form. Compositions suitable fororal administration include solid forms, such as pills, capsules,granules, tablets, and powders, and liquid forms, such as solutions,syrups, elixirs, and suspensions. Forms useful for parenteraladministration include sterile solutions, emulsions, and suspensions.

[0063] This invention is illustrated in the Experimental Details sectionwhich follows. These sections are set forth to aid in an understandingof the invention but are not intended to, and should not be construedto, limit in any way the invention as set forth in the claims whichfollow thereafter.

EXPERIMENTAL DETAILS Example 1 Legionella pneumophila is Sensitive toGemfibrozil

[0064] The original experimental objective, which led to the discoveryof a gemfibrozil-inhibitable target in bacteria, involved-the use ofgemfibrozil (GFZ) to block a eukaryotic transporter in Legionellapneumophila-infected J774 macrophages. As a control experiment, L.pneumophila was incubated with the concentration of GFZ required toinhibit the eukaryotic transporter, and it was found that growth of L.pneumophila was suppressed, which was an unexpected result. A subsequentminimum inhibitory concentration (MIC) assay demonstrated that L.pneumophila grown in AYE medium was sensitive to GFZ concentrations aslow as 10 μg/ml This was unexpected since gemfibrozil, a drug whichtherapeutically lowers triglycerides and raises HDL-cholesterol levels,has not been reported to have antimicrobial activity. The MIC assay(FIG. 1) was performed by preparing various concentrations of GFZ in AYEmedium in test tubes. L. pneumophila was added to each tube to a finalconcentration of 1×10⁶ CFUs/ml. After a 48 hour incubation at 37° C.,growth was assessed turbidimetrically (OD at 600 nm). 10 mg/ml was theminimum GFZ concentration at which no growth occurred.

[0065] MIC assays were then performed using clofibric acid, a relatedfibric acid, and probenecid, a drug which inhibits anion transporteractivity in J774 cells. Probenecid had a MIC of 160 μg/ml in AYE (FIG.2), while clofibric acid had an MIC of 125 μg/ml in AYE (FIG. 3). BothMICs were well above the 10 μg/ml seen with gemfibrozil. These resultsshowed that gemfibrozil is especially effective as an inhibitor of L.pneumophila.

[0066] To determine whether gemfibrozil is bacteriocidal orbacteriostatic, L. pneumophila was grown for 48 hours in AYE mediumcontaining varying concentrations of GFZ. Five microliter samples ofeach culture were plated on CYE agar plates. Growth was assessed after afour day incubation period at 37° C. The GFZ concentration at which nogrowth was seen on the CYE plates was 400 μg/ml. Therefore, GFZ at 10μg/ml is bacteriostatic rather than bacteriocidal. Other commonly usedantibiotics with bacteriostatic rather than bacteriocidal activityinclude chloramphenicol, the tetracyclines, erythromycin, andclindamycin.

[0067] Gemfibrozil Selectively Inhibits Bacteria That SynthesizeBranched Chain Fatty Acids

[0068] To determine whether the antimicrobial effect of gemfibrozil wasspecific for L. pneumophila, several strains of bacteria were screenedusing a zone of inhibition assay. This assay was performed by mixing 100μl of a bacterial suspension with 3 mls of F-top agar heated to 50° C.and then pouring the mixture over a suitable agar-nutrient plate. Whenthe overlay hardened, a disk containing GFZ was placed on the overlay,and the plate was incubated at the appropriate temperature until growthwas seen. A clear zone surrounding the disk, or a “zone of inhibition,”indicates that the drug on the disk inhibited bacterial growth. Ingeneral, the larger the zone of inhibition, the more potent the drug onthe species of bacteria being tested. Assessing zones of inhibition is aquick way of screening many bacterial species for susceptibility. A widevariety of bacteria were then screened. The results of these screensindicated that all susceptible bacteria had branched chain fatty acidsin their membranes, although not all bacteria with branched chain fattyacids were susceptible (FIG. 4).

[0069] The susceptibility of Mycobacterium tuberculosis, which containsvery long, branched chain mycolic acids, was especially interestinggiven the prevalence of, and the mortality associated with, thisorganism. Therefore, 21 strains of M. tuberculosis were tested,including pan-sensitive and multidrug resistant strains. Sensitivity ofM. tuberculosis to GFZ was assessed by a placing agemfibrozil-containing disk in the bottom of one of four quadrants of apetri dish. Five milliliters of Middlebrook agar were then poured overthe disk or disks in each quadrant, and the plates were incubatedovernight at room temperature to allow diffusion of the drug throughoutthe quadrant. A saline suspension containing M. tuberculosis at aMcFarland standard of two was prepared, and then diluted 10⁻². 100 μl ofthis dilution was added to each quadrant, and the plates were incubatedat 37° C. for three weeks. The presence or number of colonies in each ofthe quadrants then was assessed. The GFZ concentration at which nogrowth was seen was considered to be the MIC. All strains weresusceptible to concentrations between 100 μg/ml and 200 μg/ml ofgemfibrozil (FIG. 5). Although the inhibitory concentration is higherthan the concentration of gemfibrozil used in humans treated forhyperlipidemia (15-30 μg/ml), all 21 strains were susceptible to GFZwithin a two-fold concentration range. No greater than a two folddifference in sensitivity was seen. This suggests that none of thepresently evolved antibiotic resistance mechanisms affect sensitivity togemfibrozil, and that it has a novel target site.

[0070] Development of a Mutant of L. pneumophila with IncreasedResistance to GFZ

[0071] The discovery that L. pneumophila was sensitive to GFZnecessitated the development of a L. pneumophila-derived GFZ-resistantmutant that could be used in the transporter experiments. Efforts toobtain spontaneous mutants by plating 10⁸ wild type L. pneumophila onCYE agar plates containing GFZ were unsuccessful. Therefore, thealkylating agent ethyl methane sulfonate (EMS) was used to mutagenizethe DNA of L. pneumophila cultures. Although attempts to generate afully resistant mutant were unsuccessful, development of asemi-resistant mutant was successful. The mutant, F4b, had an MIC of 50μg/ml GFZ. Similar attempts to generate a Bacillus subtilisgemfibrozil-resistant mutant, by either spontaneous mutagenesis or byEMS mutagenesis were completely unsuccessful. The inability to develophigh-level gemfibrozil-resistant mutants in either species of bacteriasuggests that gemfibrozil's target may be an essential gene product inthese bacteria.

[0072] GFZ Induces the Accumulation and Expansion of Lipid-likeInclusion Bodies in L. pnuemophila

[0073] Since the mechanism of action and target of GFZ was stillunclear, Nile Blue A fluorescence was used to assess the morphology ofL. pneumophila grown in the presence of sub-inhibitory concentrations ofthe drug. Nile Blue A is a water soluble basic oxazine dye thatfluoresces at 460 nm. This dye has greater specificity and higheraffinity than Sudan Black for polyhydroxybutyrate (PHB) and does notstain glycogen and polyphosphate inclusions. As wild type L. pneumophilaenter stationary phase in the absence of gemfibrozil, they tend toelongate and accumulate numerous non-distending granules (FIG. 6A).However, staining of L. pneumophila grown to stationary phase in thepresence of GFZ demonstrated that there was a subpopulation of bacteriawith few to no inclusions, and a subpopulation of bacteria distended bylarge granules (FIG. 6B). The ability of Nile Blue A to stain thesegranules indicates that they are composed of PHB or other types ofpolyhydroxy alkanoic acids (PHAs).

[0074] PHAs are natural polyesters of B-hydroxyacyl monomer units, threeto fourteen carbons in length. Hydroxyacyl monomer units can be utilizedby bacteria as a carbon source, as precursors in fatty acid synthesis,or, in some bacterial species, stored as PHA in inclusion bodies. PHAforming species include Bacillus megaterium, Pseudomonas oleovorans,Psuedomonas aeruginosa, Alcaligenes eutrophus, and some Rhodococcus sp.,Corynebacterium sp., and Nocardia sp. strains. P. aeruginosa is notsusceptible to GFZ, but does form PHA granules. Therefore, the abilityto form PHA inclusions does not seem to be correlated withsusceptibility. However, the ability of some species, such as P.oleovorans, to incorporate branched chain hydroxyacyl fatty acidprecursors into PHA, suggests that the distending granules seen in L.pneumophila exposed to GFZ might be composed of branched chain fattyacid precursors. Since only bacteria that synthesize branched chainfatty acids are susceptible, it is possible that a metabolic block inbranched chain fatty acid synthesis induced by GFZ would result in theaccumulation of precursors. In bacteria capable of producing PHAinclusions, accumulation of precursors might result in their packagingand storage in PHA granules.

[0075] Experiments utilizing electron microscopy yielded confirmed thefluorescence data. Log phase L. pneumophila grown on CYE agar plateswith no gemfibrozil, contained one or two small lipid-like inclusions(FIG. 7). In contrast, L. pneumophila and L. pneumophila mutants thatwere partially resistant to GFZ, grown on CYE-GFZ drug plates, appearedas either bacilli without inclusions, or, as short, swollen bacilli(about 2× the normal diameter) packed with large inclusions (FIG. 8).These results suggested that GFZ induced the accumulation of a metabolicprecursor that was incorporated into the inclusion bodies seen insusceptible L. pneumophila.

[0076] A second EM experiment compared log phase growth of L.pneumophila and the GFZ resistant F4b mutant in AYE broth, in thepresence and absence of a sub-inhibitory concentration of gemfibrozil.The concentration of GFZ used inhibited L. pneumophila growth in AYE,but did not inhibit F4b growth in AYE. Four and one half hours after theaddition of GFZ to the log phase AYE cultures, L. pneumophila (+GFZ)(FIG. 9C) accumulated granules, while L. pneumophila (−GFZ) (FIG. 9A)did not. In contrast, there was no apparent difference in the number orsize of inclusions present in F4b in the presence (FIG. 9D) or absence(FIG. 9B) of GFZ. This experiment demonstrated an intermediate stage ininclusion accumulation in wild type L. pneumophila.

[0077] In summary, the presence of gemfibrozil induces the *accumulationof inclusions in L. pneumophila, and, induces large, distendinginclusions in a subpopulation of these susceptible bacteria.Additionally, the inclusions have a lipid-like morphology by EM, andstain with Nile Blue A, indicating that they may be composed of PHAs.These results suggest that the large, distending inclusions may be dueto the accumulation of a precursor involved in fatty acid metabolism.

[0078] Gemfibrozil Affects the Fatty Acid Composition of L. Pneumophila,but not its Semi-Resistant Derivative, F4b.

[0079] If GFZ affects enzyme(s) involved in fatty acid synthesis, andinhibition of this enzyme results in the accumulation of fatty acidprecursors, then exposure to GFZ should alter the fatty acid compositionof GFZ-susceptible bacteria. To address this possibility, gaschromatograhy was used to compare the fatty acid profiles of L.pneumophila (FIG. 10A) and the semi-resistant mutant, F4b (FIG. 10C),grown in the presence and absence of sub-inhibitory concentrations ofgemfibrozil. Base hydrolysis was used to saponify the fatty acids, whichwere then methylated, extracted, and injected into a gas chromatograph.A step temperature program was used such that as the temperatureincreased, progressively longer chain fatty acids were released from thecolumn and detected as peaks on the chromatograph. The presence ofgemfibrozil resulted in decreased peak areas for several typical L.pneumophila fatty acids, and, the appearance of several new fatty acids(FIG. 10B). This indicated that GFZ inhibited the synthesis of several“typical” fatty acids and suggested that “new” fatty acids accumulate asa result of a metabolic backup. In FIG. 10B, fatty acid peaks whichdecreased in the presence of GFZ are marked by downward arrows, newpeaks which appeared in the presence of GFZ are marked-by dots.

[0080] Importantly, the presence of gemfibrozil did not affect the fattyacid profile of F4b (FIG. 10D). This indicates that F4b resistance maybe mediated by an enzyme with a lower affinity for GFZ, and, that thisenzyme is involved in fatty acid synthesis. Additionally, the fatty acidprofile of F4b looked quite different from that of wild type L.pneumophila, in that it had fewer peaks than L. pneumophila. Although nonew peaks were detected in F4b, there were fewer peaks, and those peakswhich were present, were present in different proportions than in L.pneumophila. The identity of the fatty acids has not been determined inthe chromatograms since many of them were not present in the standard. Adisappointing limitation was the inability to detect or identify fattyacids less than twelve carbons long using this system. Identification ofshorter chain fatty acids may be useful in determining the identity ofthe gemfibrozil-induced inclusions.

[0081] Isoniazid (INH)-Resistant F4b Revertants

[0082] Since there is significant evidence supporting the hypothesisthat gemfibrozil affects fatty acid metabolism in L. pneumophila, L.pneumophila and F4b were tested for sensitivity to isoniazid, atuberculostatic drug which targets an enoyl reductase involved inmycolic fatty acid synthesis in M. tuberculosis. While wild type L.pneumophila showed no sensitivity to isoniazid, the semi-resistantmutant, F4b, was sensitive (FIG. 11). This indicated that the mutationresponsible for GFZ resistance might also have conferred isoniazidsensitivity.

[0083] To see whether isoniazid sensitivity and gemfibrozil resistancehad a reciprocal relationship (which would imply that they share thesame target enzyme) INH-resistant derivatives of F4b were isolated. Todo this, the isoniazid-sensitive, gemfibrozil-resistant, F4b strain wasplated out on CYE drug plates containing isoniazid 400 μg/ml, andscreened for spontaneous mutants resistant to isoniazid. Single coloniesof spontaneous mutants arose at a rate of 1×10⁻⁷. Several of thesecolonies were picked and purified by passage on nonselective CYE agarplates. The purified F4b derived INH-resistant strains were then testedfor both isoniazid sensitivity and gemfibrozil sensitivity using thezone of inhibition assay (FIG. 12).

[0084] The colonies indicated by the left bar under the histogrammaintained the parental, F4b, INH-sensitive GFZ-resistant phenotype,indicating that they were not true INH-resistant revertants. Since thepurification was nonselective, contaminating parental F4b bacteria mayform colonies which are picked for father passage. Also,isoniazid-resistance due to up regulation of an enzyme, rather than agenetic-mediated resistance, would be lost in the absence of a selectivepressure. Therefore, when the “purified” colonies are retested forisoniazid sensitivity, one expects to see colonies with either theparental phenotype or a genetically-mediated isoniazid-resistancephenotype.

[0085] The colonies indicated by the right bar under the histogramregained GFZ sensitivity as INH sensitivity was lost. The reciprocalrelationship between gemfibrozil sensitivity and isoniazid sensitivityin these revertants, indicates that both drugs have the same targetenzyme but different target sites. The mutation which allows gemfibrozilresistance (and probably affects substrate recognition) may result in aconformational change exposing an isoniazid sensitive site. InMycobacteria sp., the INH target, enoyl reductase, is encoded by inhA.Since GFZ appears to target an enzyme which can be made INH-sensitive,it is probably targeting an InhA homologous enzyme in L. pneumophila.

Example 2 Additional Evidence for an InhA Homologous Target: Differencesin Sensitivity to Ethionamide

[0086] Ethionamide sensitivity provided additional evidence to supportthe hypothesis that the target gene in Legionella is homologous to theInhA gene. Ethionamide is a second line anti-tuberculosis drug which isthought to target the same enzyme as isoniazid in Mycobacteria sp. IfGFZ is targeting the homologous enzyme in L. pneumophila, and F4bresistance to GFZ is mediated by a conformational change in the targetenzyme, then sensitivity to ethionamide might also differ between L.pneumophila and F4b. Using a zone of inhibition assay, wild type L.pneumophila was twice as sensitive to ethionamide as F4b (FIG. 13).

[0087] Growth of Intracellular L. pneumophila is Inhibited byGemfibrozil

[0088] Since bacterial species such as L. pneumophila and M.tuberculosis are primarily intracellular pathogens, for an antibiotic tobe effective it must affect bacterial growth within host white bloodcells. For example, the drug must permeate macrophages, and have accessto the intracellular compartment containing the pathogen. It is equallyimportant that factors or nutrients provided by the host white bloodcells do not bypass the metabolic step blocked by the drug.

[0089] GFZ at 100 μg/ml partially inhibited growth of L. pneumophila inhuman monocytes, macrophages, HL-60 cells (a human leukemic cell line),and J774 cells (a mouse macrophage cell line). In these experiments,monolayers of human or mouse cells were infected for two and one halfhours with wild type L. pneumophila or the GFZ semi-resistant Legionellamutant F4b and then washed to remove extracellular bacteria. GFZ wasadded to the medium at a concentration of 100 μg/ml, and the cells wereincubated at 37° C. Bacteria were-assayed at the specified time pointsby lysing the cells in each monolayer, combining the cell lysate withthe medium from the same well, and plating for CFU's on CYE agar plates.Since L. pneumophila and F4b do not replicate in the medium, any growthinhibition measured will be due to inhibition of intracellularreplication by GFZ. The presence of GFZ (100 μg/ml) did not affect HL-60cell viability after a 5 day period, so inhibition of intracellular L.pneumophila by GFZ is not due to decreased host cell viability.

[0090] In HL-60 cells, L. pneumophila growth was inhibited by three logsover a 54 hour time period when GFZ was present in the media at aconcentration of 100 μg/ml (FIG. 14A). Growth of F4b, the semi-resistantmutant, was only inhibited by one log by this concentration of GFZ. GFZinhibited intracellular growth of L. pneumophila and of F4b to about thesame extent as extracellular growth in AYE medium. The ability of GFZ toprotect HL-60 cells from intracellular L. pneumophila-induced lysis wasassessed using an MTT assay (FIG. 14B). This assay, which measures cellviability as a function of the ability of the monolayer to reduce MTT,showed increased viability of L. pneumophila infected HL-60 cells, inthe presence of GFZ 100 μg/ml, over a five day incubation period.

[0091] Growth of intracellular L. pneumophila in human monocytes wasinhibited by GFZ(100 μg/ml) by one log after a 72 hour incubation period(FIG. 15A). Similarly, growth of F4b in human macrophages (FIG. 15B),and of L. pneumophila in mouse J774 macrophages (FIG. 15C) was inhibitedby one log in the presence of GFZ 100 μg/ml. These experimentsdemonstrate that intracellular L. pneumophila remain sensitive to growthinhibition by GFZ.

[0092] A L. pneumophila 2.1 kb DNA Insert Complements an E.coli ts envMMutant and Confers Sensitivity to GFZ

[0093] Based on the above arguments, it is possible to hypothesize thatGFZ affects the inhA homologous gene in L. pneumophila. Since InhA fromM. tuberculosis has significant sequence similarity (40% identity over203 amino acids) to the EnvM protein of E.coli, a cloning strategy wasemployed in which a temperature sensitive envM E.coli mutant, ts100, wastransformed with DNA from a L. pneumophila library. A 2.1 kb insert ofL. pneumophila DNA, expressed from a pBluescript vector, was found tocomplement the EnvM ts phenotype. When the envM ts mutant was grown atthe permissive temperature (30° C.), with or without the insert, it wasnot sensitive to GFZ. However, when the ts envM E. coli was grown at therestrictive temperature (42° C.) on low osmolarity LB plates, the tsEnvM enzyme was nonfunctional, and growth was dependent on theexpression of the homologous L. pneumophila enzyme encoded by the 2.1 kbDNA insert. Under restrictive conditions (42° C. on low osmolarity LBplates), the ts envM E. coli strain was sensitive to GFZ indicating thatthe protein encoded by the 2.1 kb DNA is the target, or a target, of GFZ(FIG. 16).

[0094] Disks containing 5 mg GFZ were required to affect growth ofts100envM:pBsk2.1 at 42° C. Whether this indicates differences in thesubstrates and products of EnvM, in E. coli and L. pneumophila isuncertain. For example, if GFZ predominantly interferes with the abilityof the L. pneumophila enzyme to utilize branched-chain or long chainfatty acid precursors, but does not interfere with the ability of theenzyme to utilize straight chain or short chain precursors, then GFZwould be expected to have less of an effect in E. coli which synthesizesmost, if not all, of its fatty acids from B-hydroxy butyrate, a fourcarbon precursor of straight chain fatty acid synthesis. It has beenrecently demonstrated that the E.coli EnvM enzyme reduces a four carbonfatty acid crotonyl CoA substrate, while the homologous M. tuberculosisInhA enzyme will not reduce fatty acid substrates less than eightcarbons long. Although untested, the homologous enzymes in E. coli andM. tuberculosis may differ significantly in their ability to accept andreduce branched chain fatty acid precursors.

[0095] Since the 2.1 kb L. pneumophila insert confers GFZ sensitivity,the next step would be to sequence the envM homologous gene contained inthis insert. Once the gene, is sequenced it can be tagged and expressedfrom high copy plasmids to facilitate purification for biochemicalassays. Such assays may be used to directly assess in vitro inhibitionof enzyme function by GFZ. EnvM and InhA activity have been measured invitro by a NADH oxidation assay. In this assay, the purified enzyme,fatty acid CoA substrate, and NADH are combined in a cuvette, and NADHoxidation is measured over time at 340 nm in a spectrophotometer. Thisassay may be utilized to test the purified EnvM homologous enzyme. GFZmay inhibit NADH oxidation.

[0096] Once the L. pneumophila envM homologous gene is sequenced, PCRcan be used to pull out the homologous gene from the GFZ semi-resistantmutant F4b. This gene can then be transformed into wild type L.pnuemophila to see if its expression confers resistance to GFZ.Additionally the homologous protein from GFZ semi-resistant F4b can betested for resistance to GFZ biochemically.

[0097] It is possible that there is more that one enoyl reductase in L.pneumophila (E.coli contains two known enoyl reductases). The envMhomologous gene can also be used to hybridize to other potential enoylreductases in a L. pneumophila library, and potentially pull out otherGFZ sensitive targets. Once the target genes are identified,site-directed mutagenesis can be used to identify the GFZ and substratebinding sites.

[0098] Discussion

[0099] In summary, a compound, GFZ, was identified which appears toinhibit fatty acid synthesis in several species of bacteria containingbranched chain fatty acids. The GFZ target in L. pneumophila may befully characterized and utilizing both genetic and biochemicalapproaches. Once the target has been identified, site-directedmutagenesis van be used for structure-function analysis to determine itsGFZ binding site. Although the enzymatic target is found in otherorganisms beyond Mycobacteria, this enzyme has not been utilized as atarget in any other species of bacteria. GFZ appears to have a novel andessential target site on the enzyme, since cross-resistance associatedwith other antibiotics has not been seen, and no high level resistantmutants have been obtained. It is possible that bacteria that do notcontain branched chain fatty acids have a similar enzymatic site thatcan be targeted by other compounds or GFZ derivatives. Sensitivity canbe tested biochemically using the NADH oxidation assay described above.Identification of the protein targeted by gemfibrozil, and the role ofthis protein in synthesizing fatty acids from, specific precursors, andwhich enzymatic sites are important for these reactions, should beinformative for both basic biology and for medicinal therapy. Theability of GFZ to inhibit synthesis of some, but not all fatty acidprecursors in bacteria suggests it may have a similar effect ineukaryotic cells. Thus, these studies may provide insight into themechanism by which this drug lowers blood lipids in humans.

[0100] References

[0101] Brown, W. V. Potential use of fenofibrate and other fibric acidderivatives in the climic. Am. J. Med., 1987, 83, Suppl. 5B, 85-89.

[0102] Oliver, M. F.; Heady, J. A.; Morris, J. N. and Cooper, M. M. Aco-operative trial in the primary prevention of ischaemic heart diseaseusing clofibrate. Br. Heart J., 1987, 40, 1069-1118.

[0103] Palmer, R. H. Prevalence of gallstones in hyperlipidemia andincidence during treatment with clofibrate and/or cholestyramine. Trans.Assoc. Am. Physicians, 1987, 91, 0424-432.

1. A method for inhibiting growth of a bacterium which consistsessentially of contacting the bacterium with a compound having thestructure:

wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ comprises independently H, F,Cl, Br, I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇COR₈,—NO₂,—(CH₂)_(p)OR₇, —(CH₂)_(p)X(R₇)₂, —(CH₂)_(p)XR₇COR₈, a straightchain or branched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,thioalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl, orheteroaryl; wherein a linkage to the benzene ring may alternatively be—N—, —S—, —O— or —C—; wherein R₇R₈ may be independently H, F, Cl, Br, I,—OH, —CN, —COH, —SH₂, —NH₂, —NHCOH, —(CH₂)_(p)OH, —(CH₂)_(p)X(CH₂),—(CH₂)_(p)XCOH, a straight chain or branched, substituted orunsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl,phenyl, substituted phenyl, or heteroaryl; wherein A may be —N₂—, —NH—,—C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—CH₂—, —CH₂—CH₂—O —, —CH₂—CH₂—CH₂—O—, —S—,—S(═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—; and wherein Q, p, n and Xmay independently be an integer from 1 to 10, or if Q is 1 A comprises a(C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chainwhich is branched or unbranched, substituted or unsubstituted and canoptionally be interrupted 1 to 3 times by —O— or —S— or —N—; or apharmaceutically acceptable salt or ester thereof, which compound ispresent in a concentration effective to inhibit growth of the bacterium.2. The method of claim 1, wherein A comprises an (C₁-C₁₀)-alkylenechain, (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynylchain which is branched or unbranched, substituted or unsubstituted andcan optionally be interrupted 1 to 3 times by —O— or —S— or —N—.
 3. Themethod of claim 1, wherein R₁=R₄=CH₃ or —OH, R₂=R₃=R₅=R₆=H or —OH,A=CH₂, and Q=3.
 4. The method of claim 1, wherein R₃=Cl,R₁=R₂=R₄=R₅=R₆=H or —OH, and Q=0.
 5. The method of claim 1, wherein

R₆=CH(CH₃)₂, R₁=R₂=R₄=R₅=H or —OH, and Q=0.
 6. The method of claim 1,wherein R₃=Cl, R₆=C₂H₅, R₁=R₂=R₄=R₅=H or —OH, and Q=0.
 7. The method ofclaim 1, wherein the bacterium is Legionella pneumophila, Mycobacteriumtuberculosis, Bacillus subtilis, Bacillus Megaterium, PseudomonasOleovorans, Alcaligenes eutrophus, Rhodococcus sp., Citrobacter freundi,Group A Streptococcus sp., Coag neg Staphylococcus aureus or Nocardiasp.
 8. The method of claim 1, wherein the bacterium is Legionellapneumophila.
 9. The method of claim 1, wherein the bacterium isMycobacterium tuberculosis.
 10. The method of claim 1, wherein thebacterium is in a eukaryotic cell.
 11. The method of claim 1, whereinthe concentration of the compound is from about 5 μg/ml to about 100μg/ml.
 12. The method of claim 1, wherein the concentration of thecompound is 20 μg/ml.
 13. A method for alleviating the symptoms of abacterial infection in a subject which consists essentially ofadministering to the subject an amount of a compound having thestructure:

wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ may be independently H, F, Cl,Br, I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇COR₈, —NO₂,—(CH₂)_(p)OR₇, —(CH₂)_(p)X(R₇)₂, —(CH₂)_(p)XR₇COR₈, a straight chain orbranched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl,methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;wherein a linkage to the benzene ring may alternatively be —N—, —S—, —O—or —C—; wherein R₇ or R₈ may be independently H, F, Cl, Br, I, —OH, —CN,—COH, —SH₂, —NH₂, —NHCOH, —(CH₂)_(p)OH, —(CH₂)_(p)X(CH₂),—(CH₂)_(p)XCOH, a straight chain or branched, substituted orunsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl,phenyl, substituted phenyl, or heteroaryl; wherein A may be —N₂—, —NH—,—C═C═CH₂—, —C≡C—CH₂HOH—, —C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—, —S—,—S(═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—; and wherein Q, p, n and Xmay independently be an integer from 1 to 10, or if Q is 1 A may be a(C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chainwhich is branched or unbranched, substituted or unsubstituted and canoptionally be interrupted 1 to 3 times by —O— or —S— or —N—; or apharmaceutically acceptable salt or ester thereof, which compound ispresent in a concentration effective to inhibit bacterial growth andthus alleviate the symptoms of the bacterial infection in the subject.14. The method of claim 13, wherein A comprises an (C₁-C₁₀)-alkylenechain, (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynylchain which is branched or unbranched, substituted or unsubstituted andcan optionally be interrupted 1 to 3 times by —O— or —S— or —N—.
 15. Themethod of claim 13, wherein R₁=R₄=CH₃ or —OH, R₂=R₃=R₅=R₆=H or —OH,A=CH₂, and Q=3.
 16. The method of claim 13, wherein R₃=Cl,R₁=R₂=R₄=R₅=R₆=H or —OH, and Q=0.
 17. The method of claim 13, wherein

R₆=CH(CH₃)₂, R₁=R₂=R₄=R₅=H or —OH, and Q=0.
 18. The method of claim 13,wherein R₃=Cl, R₆=C₂H₅, R₁=R₂=R₄=R₅=H or —OH, and Q=0.
 19. The method ofclaim 13, wherein the bacterial infection is associated with Legionellapneumophila, Mycobacterium tuberculosis, Bacillus subtilis, BacillusMegaterium, Pseudomonas Oleovorans, Alcaligenes eutrophus, Rhodococcussp., Citrobacter freundi, Group A Streptococcus sp., Coag negStaphylococcus aureus or Nocardia sp.
 20. The method of claim 13,wherein the bacterial infection is associated with Legionellapneumophila.
 21. The method of claim 13, wherein the bacterial infectionis associated with Mycobacterium tuberculosis.
 22. The method of claim13, wherein the subject is a human or an animal.
 23. The method of claim13, wherein the bacterial infection is associated with Leprosy, Brucellaor Salmonella.
 24. The method of claim 13, wherein the concentration ofthe compound is from about 5 μg/ml blood of the subject to about 180μg/ml blood of the subject.
 25. The method of claim 13, wherein theconcentration of the compound is 90 μg/ml blood of the subject.
 26. Themethod of claim 13, wherein the administration to the subject is oral.27-40. (canceled)
 41. A method of altering a pathway of fatty acidsynthesis in a bacterium which comprises contacting the bacterium with acompound having the structure:

wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ may be independently H, F, Cl,Br, I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇COR₈, —NO₂,—(CH₂)_(p)OR₇, —(CH₂)_(p)X(R₇)₂, —(CH₂)_(p)XR₇COR₈, a straight chain orbranched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl,methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;wherein a linkage to the benzene ring may alternatively be —N—, —S—, —O—or —C—; wherein R₇ or R₈ may be independently H, F. Cl, Br, I, —OH, —CN,—COH, —SH₂, —NH₂, —NHCOH, —(CH₂)_(p)OH, —(CH₂)_(p)X(CH₂),—(CH₂)_(p)XCOH, a straight chain or branched, substituted orunsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl,phenyl, substituted phenyl, or heteroaryl; wherein A may be —N₂—, —NH—,—C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—, —S—,—S(═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—; and wherein Q, p, n and Xmay independently be an integer from 1 to 10, or if Q is 1 A may be a(C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chainwhich is branched or unbranched, substituted or unsubstituted and canoptionally be interrupted 1 to 3 times by —O— or —S— or —N—; or apharmaceutically acceptable salt or ester thereof, thus altering thepathway of fatty acid synthesis.
 42. The method of claim 41, wherein Acomprises an (C₁-C₁₀)-alkylene chain, (C₁-C₁₀)-alkyl chain,(C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chain which is branched orunbranched, substituted or unsubstituted and can optionally beinterrupted 1 to 3 times by —O— or —S— or —N—.
 43. The method of claim41, wherein R₁=R₄=CH₃ or —OH, R₂=R₃=R₅=R₆=H or —OH, A=CH₂, and Q=3. 44.The method of claim 41, wherein R₃=Cl, R₁=R₂=R₄=R₅=R₆=H or —OH, and Q=0.45. The method of claim 41, wherein

R₆=CH(CH₃)₂, R₁=R₂=R₄=R₅=H or —OH, and Q=0.
 46. The method of claim 41,wherein the bacterium is Legionella pneumophila, Mycobacteriumtuberculosis, Bacillus subtilis, Bacillus Megaterium, PseudomonasOleovorans, Alcaligenes eutrophus, Rhodococcus sp., Citrobacter freundi,Group A Streptococcus sp., Coag. neg Staphylococcus aureus or Nocardiasp.
 47. A method of inhibiting growth of a bacterium which consistsessentially of contacting the bacteria with an enoyl reductase inhibitorso as to inhibit the reductase and thus inhibit the growth of thebacterium.
 48. A method for determining whether or not a bacterium issensitive to a compound having the structure:

wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ may be independently H, F, Cl,Br, I, —OH, —OR₇, —CN, —COR₇, —SR, —N(R₇)₂, —NR₇COR₈, —NO₂,—(CH₂)_(p)OR₇, —(CH₂)_(p)X(R₇)₂, —(CH₂)_(p)XR₇COR₈, a straight chain orbranched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl,methylene thioalkyl, acyl, phenyl, substituted phenyl, or heteroaryl;wherein a linkage to the benzene ring may alternatively be —N—, —S—, —O—or —C—; wherein R₇ or R₈ may be independently H, F, Cl, Br, I, —OH, —CN,—COH, —SH₂, —NH₂, —NHCOH, —(CH₂)_(p)OH, —(CH₂)_(p)X(CH₂),—(CH₂)_(p)XCOH, a straight chain or branched, substituted orunsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl, acyl,phenyl, substituted phenyl, or heteroaryl; wherein A may be —N₂—, —NH—,—C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—, —S—,—S(═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—; and wherein Q, p, n and Xmay independently be an integer from 1 to 10, or if Q is 1 A may be a(C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or (C₁-C₁₀)-alkynyl chainwhich is branched or unbranched, substituted or unsubstituted and canoptionally be interrupted 1 to 3 times by —O— or —S— or —N—; or apharmaceutically acceptable salt or ester thereof, which comprisescontacting the bacterium with a concentration of the compound effectiveto inhibit growth of the bacterium if the bacterium is sensitive to thecompound, thereby determining whether or not the bacterium is sensitiveto the compound.
 49. The method of claim 48, wherein A comprises an(C₁-C₁₀)-alkylene chain, (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or(C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted orunsubstituted and can optionally be interrupted 1 to 3 times by —O— or—S— or —N—.
 50. The method of claim 48, wherein R₁=R₄=CH₃, R₂=R₃=R₅=R₆=Hor —OH, A=CH₂ or —OH, and Q=3.
 51. The method of claim 48, whereinR₃=Cl, R₁=R₂=R₄=R₅=R₆=H or —OH, and Q=0.
 52. The method of claim 48,wherein

R₆=CH(CH₃)₂, R₁=R₂=R₄=R₅=H or —OH, and Q=0.
 53. The method of claim 48,wherein R₃=Cl, R₆=C₂H₅, R₁=R₂=R₄=R₅=H or —OH, and Q=0.
 54. The method ofclaim 48, wherein the bacterium is in a cell.
 55. The method of claim48, wherein the bacterium is selected from the group consisting ofLegionella pneumophila, Bacillus subtilis, Caulobacter crescentus,Citrobacter freundi, Nocardia sp., Rhodobacter spheroides, Group A.Streptococcus sp., Coag neg Staphylococcus aureus and Mycobacteriumtuberculosis.
 56. The method of claim 48, wherein the concentration ofthe compound is from about 5 μg/ml to about 100 μg/ml.
 57. The method ofclaim 48, wherein the concentration of the compound is 20 μg/ml. 58-59.(canceled)